分离自Kefir的植物乳杆菌体外降胆固醇作用的研究

对分离Kefir中的植物乳杆菌体外降胆固醇作用进行了研究。结果表明，植物乳杆菌在含胆盐的培养基中具有良好的降胆固醇能力，24h后培养基中的胆固醇可降低60％(0．18g／L)；该菌体外降胆固醇作用的机理主要是其产生的胆盐水解酶水解胆盐变为游离态的胆酸，与胆固醇形成复合物，在酸性pH值条件下发生共沉淀。同时，也发现该菌产生的胆盐水解酶对反应底物(各种胆盐)的特异性不同，以脱氧牛磺胆酸最佳。     

Short communication: Improving the activity of bile salt hydrolases in Lactobacillus casei based on in silico molecular docking and heterologous expression

Bile salt hydrolase (BSH) plays an essential role in the cholesterol-removing effect of lactic acid bacteria, which hydrolyze conjugated bile salts to amino acid and deconjugated bile salts. However, Lactobacillus casei lacks the bsh gene, which may make it highly sensitive to bile salt stress. We wanted to improve the BSH activity of L. casei for various food-industry applications (e.g., milk fermentation). Plate assay testing indicated that Lactobacillus plantarum AR113 has the highest BSH activity. We cloned and sequenced 4 bsh genes from the genome of L. plantarum AR113. Structure modeling and molecular docking of BSH indicated that BSH1 and BSH3 could react efficiently with bile salts, so we selected BSH1 and BSH3 for heterologous expression in L. casei. Compared with single expression of BSH1 or BSH3, co-expression of both protein sequences showed the highest hydrolysis activity by HPLC analysis. Our results suggested that heterologous expression of BSH in L. casei can significantly improve host activity against bile salts, and in silico molecular docking could be an efficient method of rapid screening for BSH with high activity.     

Construction of R16F and D19L mutations in the loop I of bile salt hydrolase (BSH) enzyme from Lactobacillus plantarum B14 and structural and functional analysis of the mutant BSHs

Bile salt hydrolase (BSH), found commonly in intestinal species of Lactobacillus and Bifidobacterium, catalyzes the hydrolysis of glycine or taurine-conjugated bile acids into the amino acids and free bile acids. Deconjugated bile acids potentially play an important role in the reduction of blood cholesterol level and formation of some gastrointestinal diseases such as cholestasis, gallstone formation, and colon cancer. Although the crystal and three-dimensional structures of BSH enzyme are known, the working mechanism of catalytic activity of such an important BSH enzyme is not known very well. Previous in silico analysis of multiple BSH has identified that Arginine-16 (R16) and Aspartate-19 (D19) were catalytically important residues in the active site of BSH. To confirm the function of these amino acids, in this study, BSH enzyme from Lactobacillus plantarum B14 strain was cloned into Escherichia coli and strictly conserved polar R16 and D19 amino acids of BSH enzyme were substituted for hydrophobic Phenylalanine-16 (F16) and Leucine-19 (L19) amino acids, respectively, by polymerase chain reaction (PCR)-based site-directed mutagenesis. The effects of the mutations on catalytic activity and structure of the BSH enzymes were detected by ninhidrin assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, respectively. Research results showed that although F16 mutation led to loss of enzyme activity completely, L19 mutation led to abolishment of the synthesis of BSH enzyme. These results indicated that R16 and D19 amino acids located in loop I of the BSH enzyme might be critical for catalytic activity and assembly of the BSH enzyme respectively.     

一株乳酸乳球菌的降胆固醇特性及其作用机制

探讨了乳酸乳球菌乳亚种HUCM 201的降胆固醇特性及其体外去除胆固醇的机制。乳酸乳球菌乳亚种HUCM 201菌株可从培养基中去除33.1%的胆固醇,其中14.3%的胆固醇发生共沉淀并重新溶解在洗涤液中,18.1%的胆固醇被吸收到菌体细胞内。HUCM 201菌株对5种结合型胆酸盐分别表现出了不同的胆盐水解酶活性,其中对甘氨胆酸钠的水解能力最强,总酶活为0.279 U/mL,比酶活为0.076 U/mg。     

Bile salt deconjugation ability,bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains

Eleven strains of lactobacilli were screened for their bile salt deconjugation ability, bile salt hydrolase activity (BSH) and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation as determined by the release of cholic acid showed that more cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate, and Lactobacillus acidophilus strains had higher deconjugation ability than L. casei strains. BSH activity, as quantified by the amount of taurine or glycine liberated from conjugated bile salts, indicated that substrate specificity was more towards glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both conjugated bile, with more cholesterol being precipitated upon deconjugation of sodium glycocholate than upon that of sodium taurocholate. Cholesterol co-precipitation with deconjugated bile increased with decreasing pH. L. acidophilus ATCC 33200, 4356 and 4962 and L. casei ASCC 1521 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be promising candidates to exert beneficial bile deconjugation activity in vivo.     

Molecular cloning,expression and characterization of bile salt hydrolase from Lactobacillus rhamnosus E9 strain

Bile salt hydrolase (BSH) enzyme, commonly found in probiotic bacteria of gut origin, catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts allowing for colonization of the bacteria in the gut and contributing to a decrease in levels of cholesterol. However an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid can have harmful side-effects. The aim of this study was to characterize the activity of BSH enzymes from Lactobacillus rhamnosus E9, a popular probiotic strain. The bsh gene was cloned, expressed, purified and characterized in Escherichia coli BLR(DE3) strain. The hydrolysis activities and substrate specificities of the recombinant BSH (rBSH) enzyme were examined using six different bile acids. Nucleotide sequence analysis results indicated that the bsh of E9 contained an open reading frame (ORF) of 1014 and nucleotides encoding a 338-amino acid protein with a molecular weight of 37 kDa. Five catalytically important amino acids and the amino acid motifs located around the active site were highly conserved. The rBSH showed a slight preference towards glycine-conjugated to tauro-conjugated bile salts. This confirms that it is a safe strain for probiotics and its preference for glycine-conjugated bile salts should be further investigated.     

4种胆盐水解酶在发酵乳杆菌AR497中的异源表达

为提高发酵乳杆菌耐胆盐能力,将植物乳杆菌AR113来源的4种BSH基因(BSH1、BSH2、BSH3和BSH4)转化至发酵乳杆菌AR497中进行表达。结果表明,植物乳杆菌来源的4种BSH同功酶异源表达可提高发酵乳杆菌的耐胆盐能力。在2.0mg/mL甘氨脱氧胆酸钠浓度下,表达BSH2的工程菌致死率最低,其他菌株生长被完全抑制;BSH酶活测定表明,BSH2表达菌株酶活最高,达57.74U/mL,比对照空质粒菌株提高了2.88倍。     

Bile salt deconjugation and BSH activity of five bifidobacterial strains and their cholesterol co-precipitating properties

Five strains of bifidobacteria were screened for their bile salt deconjugation ability, bile salt hydrolase (BSH) activity and co-precipitation of cholesterol with deconjugated bile. Bile salt deconjugation was determined by the release of cholic acid. All strains exhibited deconjugation of both sodium glycocholate and sodium taurocholate. More cholic acid was liberated from the deconjugation of sodium glycocholate than sodium taurocholate. BSH activity was quantified by determining the amount of glycine or taurine liberated from conjugated bile salts by bifidobacteria strains. There was higher substrate specificity for glycine-conjugated bile compared to taurine-conjugated bile. Co-precipitation of cholesterol with cholic acid was observed from deconjugation of both sodium glycocholate and sodium taurocholate, and by all bifidobacteria strains studied. More cholesterol was precipitated with cholic acid when sodium glycocholate was used compared to sodium taurocholate. Increased cholesterol co-precipitation with deconjugated bile was observed with decreasing pH levels. Bifidobacterium infantis 17930 showed highest deconjugation ability and BSH activity towards bile mixtures that resemble the human bile, and may be a promising candidate to exert beneficial bile deconjugation activity in vivo.     

具有胆盐水解酶活性乳酸菌的筛选及影响其活性因素分析

具有胆盐水解酶活性的乳酸菌有潜在的降低血液胆固醇的作用。对分离自酸马奶的10株乳杆菌的胆盐水解酶活性进行了定性及定量分析,在此基础上对影响胆盐水解酶的一些因素进行了研究。结果表明,3株乳杆菌具有胆盐水解酶活性,其中以MG13-3为最高,其次为XB4-1及MG16-3;当pH值为6时,超声波破碎时间在12～15min之间,培养基中加入巯基乙酸钠并在厌氧条件下培养时,3株乳杆菌均体现出较高的胆盐水解酶活性。     

不同来源胆盐水解酶基因在大肠杆菌中的表达

根据GenBank上报道的两歧双歧杆菌ATCC 29521的胆盐水解酶基因(BSH)序列和嗜酸乳杆菌NCFM的胆盐水解酶基因(BSHA和BSHB)序列,设计引物通过PCR扩增获得BSH基因,将其连接到表达载体pET28a(+),构建pETBSH表达质粒,经IPTG诱导表达后,聚丙烯酰胺凝胶电泳(SDS-PAGE)检测表明在分子质量大小约40、43、45kD处有预期条带出现,初步表明蛋白表达成功。重组菌产生的胆盐水解酶水解甘氨胆酸钠产生的甘氨酸经茚三酮比色法分析,表明该重组的胆盐水解酶具有水解活性。